Zona incerta (ZI) of the diencephalon has extensive connections with other brain regions and has been involved in several functions such as controlling visceral activity, thermoregulation, arousal and locomotor activity. In spite of its important functions it is one of the most understudied regions of the brain and no studies of the cellular and synaptic electrophysiological properties of ZI neurons have been reported so far. Maintenance of body temperature is central to energy homeostasis in mammals because it represents a major part of the energy expenditure of the organism. It is controlled by neurons of the preoptic area/ anterior hypothalamus (PO/AH) that project directly or indirectly to the rostral raphe pallidus. Anatomical and functional evidence clearly indicate a projection from the preoptic area, the thermoregulatory center, to ZI neurons. Combined retrograde immunostaining experiments have revealed that preoptic neurons projecting to ZI are peptidergic. ZI neurons project to the raphe pallidus and thus they can represent a relay of the preoptic thermogenic output to the brainstem neurons controlling thermoeffector processes. Our overarching hypothesis is that ZI GABAergic neurons mediate the peptidergic modulation of thermoregulation by preoptic neurons. Preliminary data indicate that ZI neurons express receptors for these neuropeptides. We have also found that thermoregulatory neuropeptides excites ZI GABAergic neurons by activating inward currents and that they increase intracellular Ca concentrations. Our preliminary data also show that preoptic neurons projecting to ZI GABAergic neurons are peptidergic. We plan to identify both GABAergic and glutamatergic ZI neurons in slices and study their firing and synaptic characteristics as well as their modulation by neuropeptides involved in thermoregulation, followed by single cell RT/PCR analysis to determine the expression patterns for these receptors (Sp. aim1). A set of experiments will utilize retrograde labeling from the raphe pallidus to identify ZI neurons projecting to that area. By employing pharmacological tools, Ca imaging, electrophysiology and single cell RT-PCR we will then study the signaling pathways and the ion conductances involved in the neuropeptides' actions on ZI neurons (Sp. Aim2). In Sp Aim 3 we will determine using telemetry and local infusions of pharmacological agents the role of AVP and neurotensin signaling in the ZIv in thermoregulation. Studying the cellular mechanisms and signaling pathways activated by thermoregulatory peptides in ZI neurons may provide central pharmacological targets for the development of hypothermic agents and for the control of energy homeostasis. In summary in this study we will address fundamental and unexplored aspects of neurophysiology and start to lay the groundwork for understanding the role of perturbed peptidergic signaling to disease.